An increasing number of polypeptides, including enzymes and non-enzymatic proteins, are being produced industrially, for use in various industries, household, food/feed, cosmetics, medicine etc. The major source for these proteins is and have been microorganism found in nature. However, since science have developed many new techniques for creating variants of polypeptides through protein engineering, it is becoming increasingly important to be able to make huge populations of protein variants, to screen and select for new properties.
The classic approach for finding polypeptides with new and special properties, have been to screen wild type organism present in nature. This has been a very successful way of generating polypeptides to be used in such diverse areas as the above mentioned industries. Furthermore it is possible to generate new variants of a protein by classical mutation of the microorganism. However, since this approach is a very labour and time consuming process, researchers have in the last two decades been developing improvements on existing polypeptides by creating artificial diversity, using more specific and therefor faster techniques, such as protein and genetic engineering.
Such artificial diversity can be generated by using recombinant DNA techniques such as site-directed or random mutagenesis. This approach generates a population of polypeptide variants which can be selected according to the newly acquired phenotype of the given organism used for the experiments. Such phenotypic screen has its limitations according to the number of mutants it would be possible to screen. First of all one would have to set up a screening assay where a given new property of a polypeptide can be detected and the given mutant can be isolated. Then the DNA encoding the polypeptide is to be isolated and characterized.
These techniques have been used successfully to create new important polypeptides exhibiting improved properties, such as higher specific activity, higher stability under high or low pH, temperature stability, stability under oxidizing conditions etc.
However successful this approach has been, there is still a limitation in the number of polypeptides it is possible to screen and test by using these techniques. Therefore it is of interest to create novel systems, whereby it is possible to combine the screening and the selection in one procedure.
Furthermore such a procedure should make it possible to pick out only those variants with the best properties under given conditions, and these variants should be picked out of a variant population consisting of more than 10.sup.8 members.
One promising approach for making such artificial diversity and selecting for a new or improved specific polypeptide feature is the technique known as "phage display". This technique couples the genotype with the phenotype making it possible to select the two characteristics together. Hereby eliminating a couple of steps present in the above mentioned approaches.
Phage display is a fairly new technique first used and described by George P. Smith in 1988 (a first attempt was made in 1985). The earliest patents were granted to R. C. Ladner (U.S. Pat. Nos. 5,096,815 and 5,223,409) relating to "Generation and selection of novel DNA-binding proteins and polypeptides" and "Directed evolution of novel binding proteins", respectively. These two patents contain a wealth of references giving the background for the techniques.
Bacteriophage display systems have been developed that link a polypeptide or peptide of interest to the DNA that encodes it. Such display systems have been used to screen peptide libraries for binding to selected target molecules and to display functional proteins with the potential of screening these proteins for desired properties (see references 12 to 15).
Recently, improvements of the display approach have made it possible to express enzymes as well as antibody fragments on the bacteriophage surface thus allowing for selection of specific properties by selecting with specific ligands (see references 2-6, and 16-18).
For display of antibodies there is a large amount of references describing this successful approach. When it comes to the display of enzymes this is a less developed area, however there are some examples of this approach (see references 1, 8, and 10).
Especially the selection principles for enzyme phages are under evaluation and development. It has been shown that the enzymatic mechanism of a given enzyme can be used as means of binding principal by the use of suicidal inhibitors (see references 1 and 9).